This application is a 371 of PCT/JP00/02051 filed Mar. 30, 2000, which claims priority to Japan 898,34/1999 filed Mar. 30, 1999.
The present invention relates to a method for fractionating and a process for producing a 7S globulin-rich fraction and an 11S globulin-rich fraction from a soybean protein-containing solution.
Soybean storage protein is precipitated at about pH 4.5 and can be relatively easily separated from components other than the protein. This is referred to as isolated soybean protein and, in many cases, soybean protein in this form is utilized in the food industry. The protein is further divided into 2S, 7S, 11S and 15S globulins according to sedimentation constants in ultracentrifugation analysis. Among them, 7S globulin and 11S globulin are predominant constituent protein components of the globulin fractions (note: 7S globulin and 11S globulin are classification names in a sedimentation method and substantially correspond to xcex2-conglycinin and glycinin according to immunological nomenclature, respectively), and both of them have specific different properties such as viscosity, coagulability, surface activity, etc. Then, factionation of 7S globulin and 11S globulin makes it possible to utilize properties of respective protein components, and it is expected to expand industrial utilization of proteins.
7S Globulin and 11S globulin are composed of several subunits. 7S Globulin is composed of three subunits, i.e., xcex1, xcex1xe2x80x2 and xcex2 subunits. 11S Globulin is composed of several subunits each of which is a pair of an acidic polypeptide (A) and a basic polypeptide (B). The molecular weights and charge states of 7S globulin and 11S globulin are very similar to each other. In particular, both globulins are diversified due to combinations of subunits, and properties thereof range to some extent to thereby overlap each other. Then, for fractionating both globulins effectively, a certain essential difference should be found out.
Known fractionation methods are as follows. That is, a method utilizing a difference in isoelectric point: extraction is carried out by adjusting pH to about the isoelectric point of 11S globulin and only 7S globulin is extracted (JP 55-124457 A); a method utilizing a difference in reactivity with calcium: a small a-mount of a calcium salt is added upon extraction to extract a 7S globulin-rich fraction (JP 48-56843 A); a method utilizing a difference in solubility at a certain pH and ionic strength: an insoluble fraction is removed in the presence of sodium chloride or potassium chloride at pH 1.2 to 4.0 to prepare 7S protein (JP 49-31843 A), or a slurry precipitated at an isoelectric point is adjusted to pH 5.0 to 5.6 and its molar concentration of sodium chloride is adjusted to 0.01 to 2 M to separate 7S and 11S fractions (JP 58-36345 A); and a method utilizing a cold-precipitation phenomenon and a reducing agent, etc.: this utilizes a phenomenon that the solubility of 11S globulin is lowered at a low temperature (referred to as cryo-precipitation phenomenon), and a soybean protein raw material is treated in the presence of a sulfurous acid compound, glutathione compound or cysteine compound in an aqueous system at pH 6.5 or higher, followed by adjusting pH to 5.5 to 7.0 and a temperature to 20xc2x0 C. or lower to fractionate into a 7S globulin-rich fraction and an 11S globulin-rich fraction (JP 61-187755 A).
These known fractionation methods skillfully utilize a difference in solubility between 7S globulin and 11S globulin due to pH, ionic strength, the presence of a certain salt, temperature, etc. However, there are such problems that these known fractionation methods are unsuitable for an industrially applicable fractionation method because clear fractionation of both globulins cannot be achieved due to overlap of their properties as mentioned above or, even if clear fractionation can be achieved to some extent, these methods are still those for the level of experimental use. Thus, problems still remain in practice. For example, in the method of JP 61-187755 A, cryo-precipitation phenomenon highly depends on a temperature and it is necessary to cool a reaction mixture to about 5xc2x0 C., which results in such a practical problem that a large amount of a sulfurous acid compound, etc. should be added to separate fractions with an industrially available low centrifugal force, as well as which results in such a problem of fractionation precision that a little amount of 11S globulin is contaminated in a soluble fraction.
Then, it has been desired to develop a fractionation method which can simply and efficiently produce a 7S globulin-rich fraction and an 11S globulin-rich fraction in an industrial scale with minimizing contamination of the soluble fraction with the insoluble fraction or vice versa.
On the other hand, phytic acid is an organic phosphate compound (myo-inositol hexakis-phosphate: 6 phosphoric acid groups are attached to inositol) which is predominantly present in plant seeds in the form of its calcium salt, magnesium salt and potassium salt. Soybeans contain about 1% of phosphorus most of which is present in the form of phytin. Since phytic acid forms a slightly soluble compound by being attached to a nutritiously important mineral component (calcium, magnesium, iron, zinc, etc.) through a chelate bond, it is pointed out that phytic acid lowers absorption of these trace minerals in a living body. In addition, phytic acid tends to form a complex with a protein and a multivalent metal cation and, normally, soybean protein contains 1 to 3% by weight of phytic acid based on the weight of the protein. An activity of decomposing phytic acid used herein refers to the activity for liberating phosphoric acid from phytic acid. A representative enzyme having such an activity is phytase.
Utilization of phytase which has an activity of decomposing phytic acid of soybean protein is divided into that for removing phytic acid which is considered to be an inhibitor of mineral absorption and that for recovering a protein having a high solubility under acidic conditions (pH 5 or lower). Examples of the former include those described in JP 49-7300 A, JP 50-130800 A and JP 4-503002 A. Examples of the latter include a method for isolating a soluble protein fraction from a phytic acid-containing protein material which comprises adding phytase to an aqueous suspension of a phytin-containing soybean protein material to decompose phytin, adjusting the suspension to pH about 4.6 to form an insoluble precipitate, collecting a protein solution, adjusting the protein solution to pH about 5.0 to 5.4 to precipitate a protein fraction and collecting the precipitated protein fraction (JP 48-18450 A); and a method for collecting a protein from a vegetable protein raw material which comprises washing the vegetable protein raw material with water at an isoelectric point, digesting the washed vegetable protein raw material with acidic phytase, and separating a soluble protein-containing liquid extract from an insoluble digested residue to collect the protein (JP 51-125300 A).
Phytase is reacted under the following conditions (extracted from Examples of each publication). JP 49-7300 A: endogenous phytase, pH 5, 65xc2x0 C., 9.3 hours; JP 50-130800 A: wheat phytase, pH 5.5, 45xc2x0 C., 16 hours; JP 4-503002 A: microorganism phytase, pH 5.0, 40xc2x0 C., 4 hours; JP 48-18450 A: wheat phytase, pH 6, 50-55xc2x0 C., 24 hours; and JP 51-125300 A: microorganism phytase, pH 2.8, 50xc2x0 C., 10 hours.
Since bacteria generally tend to grow at pH 5 or higher, normally, a solution containing soybean protein is liable to be putrefied unless it is subjected to sterilization treatment such as heat sterilization, etc. In addition, since a protein is liable to be denatured with an acid by treatment under strong acidic conditions such as at pH 2.8 for a long period of time, such treatment adversely affects fractionation of 7S globulin and 11S globulin. Further, in the method of JP 51-125300 A, the dissolved fraction is fractionated from xe2x80x9cokara (insoluble residue from soybean milk or tofu production)xe2x80x9d component after treatment with phytase. Therefore, this does not teach acceleration of fractionation of 7S globulin and 11S globulin.
An object of the present invention is to propose a novel fractionation method and a production process of 7S globulin and 11S globulin, wherein an enzyme is utilized. Another object of the present invention is to provide a fractionation method having high fractionation precision with minimizing a contamination rate of respective fractions, which can be simply and efficiently carried out the production of both fractions in an industrial scale.
As a result of the present inventors"" intensive study, the following have been found.
It has been found that fractionation capability of 7S globulin and 11S globulin is improved by treatment with phytase, which is an enzyme having an activity of decomposing phytic acid, at a certain pH. Further, it has also been found that, when a soybean protein-containing solution is treated with an enzyme or an enzyme preparation having an activity of decomposing phytic acid to decompose phytic acid and phytates contained therein, followed by separating them at an appropriate pH range, a 7S-rich fraction can be simply and readily passed into a soluble fraction, while passing an 11S-rich fraction into an insoluble fraction without overlapping to each other at room temperature without addition of a reducing agent, etc. Thus the present invention has been completed. Although the mechanism of this phenomenon is not fully elucidated, it is considered that phytic acid and phytates, which form a complex with a protein and a multivalent metal ion, are decomposed by treatment with phytase to cause change in solubility behavior of 7S globulin and 11S globulin, thereby increasing a difference in their solubility at a specific pH, which make their fractionation possible.
The present invention is a method for fractionation or a process for producing a 7S globulin-rich fraction and an 11S globulin-rich fraction which comprises treating a soybean protein-containing solution with an enzyme or an enzyme preparation having an activity of decomposing phytic acid and separating it into a soluble fraction and an insoluble fraction at a specific pH.
Hereinafter, preferred embodiments of the present invention will be described. Soybean protein used in the present invention is preferably that from which insoluble residue (xe2x80x9cokaraxe2x80x9d) has been removed. Examples thereof include soybean protein such as soybean milk (including dried soybean milk powder), isolated soybean protein and the like, whose protein is native or with little denaturation, that is, soybean protein which is processed lightly without denaturation, or with minimizing denaturation. In general, defatted-soybeans obtained by extraction with n-hexane at a low temperature are suitable for a starting material. In particular, defatted-soybeans, which are scarcely denatured and have the nitrogen solubility index (NSI) of 60 or more, preferably 80 or more, are preferred. Preferably, defatted-soybean milk and isolated soybean protein obtained from such scarcely denatured defatted-soybeans are used in the present invention.
In addition, it is preferred to avoid protein denaturation due to heating and severe conditions (e.g., strong acidic or strong alkaline conditions, etc.) as much as possible during production steps (e.g., extraction step, step for adjusting pH, etc.) thereof. Preferably, a heat sterilization step is also avoided. Normally, the phytic acid content of a solution containing thus-obtained soybean protein without denaturation or with little denaturation is 1 to 3% by weight based on the weight of the protein.
Whether protein in a soybean protein-containing solution is denatured with heat, etc. or not can be judged by analysis of the protein with Differential Scanning Calorimetry (DSC) (Nagano et al., J. Agric. Food Chem., 40, 941-944 (1992)). According to this analysis, for example, respective endothermic peaks derived from its main constituent components, 7S and 11S globulins, can be recognized in case of isolated soybean protein which is not denatured. However, when isolated soybean protein undergoes excess denaturation, no endothermic peak derived from the constituent components is observed. Therefore, presence or absence of denaturation can be readily judged.
The enzyme or the enzyme preparation having an activity of decomposing phytic acid to be used in the present invention is not specifically limited and there can be used enzymes such as phytase, phosphatase, etc. which have an activity of decomposing phytic acid and derived from vegetables such as wheat, potato, etc.; animal organs such as the intestinal tracts, etc.; and microorganisms such as bacteria, yeast, mold, actinomycetes, etc. Preferably, the enzyme or the enzyme preparation to be used has no or less protease activity because the protease activity causes not only interference with fractionation of 7S globulin and 11S globulin due to hydrolysis thereof to change their solubility behavior, but also difficulties in recovery of 7S globulin and 11S globulin as proteins. For example, in case that there is no or less protein hydrolysis with the protease activity, a TCA solubilization degree of soybean protein after treatment of the enzyme or the enzyme preparation can be defined as 20% or less, preferably 15% or less. The term xe2x80x9cTCA solubilization degreexe2x80x9d used herein is a ratio of the protein solubilized with 0.22 M trichloroacetic acid (TCA) to the total protein measured by a protein determination method such as Kjeldahl method, Lowry method, etc. In general, an activity of decomposing of phytic acid of the enzyme or the enzyme preparation derived from bacteria is higher than that derived from vegetables, and the protease activity of the former is lower than the latter. Therefore, the former is advantageous in view of prevention of hydrolysis and putrefaction of protein.
For practicing the present invention, it is necessary to treat a soybean protein-containing solution with the enzyme or the enzyme preparation having an activity of decomposing phytic acid to decomposed phytic acid and phytates contained therein in order to fractionate into a 7S globulin-rich fraction and an 11S globulin-rich fraction.
Although the degree of decomposition of phytic acid and phytates is not specifically limited, for example, preferably, phytic acid content is reduced by 50% or more in comparison with that before treatment with the enzyme or the enzyme preparation. Therefore, the conditions of treatment with the enzyme or the enzyme preparation are not specifically limited in so far as the above conditions are satisfied and optimal conditions of each enzyme or enzyme preparation can be employed. Further, a treatment method is not specifically limited. However, in order to avoid denaturation of protein due to treatment under severe conditions and putrefaction due to treatment for a long period of time, treatment time is preferably 5 minutes to 3 hours. When a certain measure for avoiding denaturation and putrefaction of protein is employed, treatment can also be carried out under conditions other than the above. For example, defatted-soybean with little denaturation is extracted with water and separated into a water insoluble fraction (xe2x80x9cokaraxe2x80x9d) and a water soluble fraction (soybean milk), and the water soluble fraction is subjected to decomposition of phytic acid at pH 3.5 to 9.0 and at a temperature of 20 to 70xc2x0 C. The enzyme or the enzyme preparation having an activity of decomposing phytic acid may be added to the water soluble fraction after adjusting pH of the fraction to 3.5 to 9.0. The amount of the enzyme or the enzyme preparation to be added ranges 0.1 to 100 unit/g, preferably 0.5 to 50 unit/g and, normally, treatment is continued for 5 minutes to 3 hours. Where treatment for a shorter period of time is desired, treatment can be carried out with the enzyme or the enzyme preparation having a higher unit. One unit of phytase activity used herein is expressed by the amount of the enzyme which can liberate 1 xcexcmol of phosphoric acid from the substrate, phytic acid, during 1 minute in an initial stage of the reaction under conditions at pH 5.5 and at 37xc2x0 C.
The degree of decomposition of phytic acid and phytates used herein was determined by measuring the amount of phytic acid in the solution directly. The amount of phytic acid was measured according to the method of Alii Mohamed (Cereal Chemistry, 63, 475, 1980).
When the soybean protein-containing solution after treatment of the enzyme or the enzyme preparation having an activity of decomposing phytic acid is adjusted to pH 5.6 to 6.6, preferably, pH 5.8 to 6.4, a 7S globulin-rich fraction and an 11S globulin-rich fraction can be readily separated. Since this process can be carried out regardless of separation temperature and addition of a reducing agent, separation can be carried out without a cooling step (cold precipitation) and addition of a sulfurous acid compound, glutathione compound or cysteine compound as described in JP 61-187755 A, and this is therefore an industrially effective process. However, contamination of an insoluble fraction with 7S globulin increases when pH is  less than 5.6, or contamination of a soluble fraction with 11S globulin increases when pH is  greater than 6.6. Then, no desired fractionation is expected. Preferably, when a soybean protein-containing solution is treated with the enzyme or the enzyme preparation at pH 5.6 to 6.6, more preferably pH 5.8 to 6.4, separation can be carried out more efficiently because re-adjustment of pH is not required.
Separation can be readily carried out by a known separation means (e.g., filtration, centrifugation, etc.), in particular, a continuous centrifugal separator (e.g., decanter) or the like. Of course, a non-continuous centrifugal separator such as a batch-wise one can also be used.
The state of fractionation of the 7S globulin-rich fraction and the 11S globulin-rich fraction according to the present invention can be evaluated based on a pattern obtained by SDS-polyacrylamide gel electrophoresis. In addition, for numerical expression of the amounts of 7S globulin and 11S globulin present in the insoluble and soluble fractions, respectively, their amounts were calculated based on the recovery rates of proteins in the insoluble and soluble fractions and the ratio of areas obtained by densitometry of the pattern obtained by SDS-polyacrylamide gel electrophoresis. 7S Globulin referred to herein is the total amount of xcex1, xcex1xe2x80x2 and xcex2 subunits and the 11S globulin content is the total amount of the acidic polypeptide (A) and the basic polypeptide (B).
After separation, the soluble fraction and the insoluble fraction can be used as such, or can further be subjected to concentration, neutralization, or drying to use as a 7S globulin-rich fraction and an 11S globulin-rich fraction. For concentration, preferably, a precipitated curd is separated and recovered by isoelectric point precipitation of the soluble fraction (pH 4.5-5.3, preferably pH 4.7-5.1) from the viewpoint of improvement of physical properties. Further, after isoelectric point precipitation, the curd can also be subjected to neutralization, heat sterilization treatment or, further, treatment with an enzyme such as a protease, etc. a sterilized, powdered form is a most common product form. Heat sterilization treatment can also be carried out by known HTST treatment, UHT treatment and the like.